Post-transcriptional regulation of gene expression is a ribonucleoprotein (RNP)-driven process, which involves RNA binding proteins (RBPs) and noncoding RNAs that regulate splicing, nuclear export, subcellular localization, mRNA stability and translation. mRNAs encoding proteins that function in a particular cell process or pathway can be found within a unique mRNP complex, which consists of mRNA and RNP. This provides valuable information regarding not only known components of a particular process or pathway, but importantly, leads to the identification of novel components representing potential therapeutic targets and biomarkers. In addition to those targets identified by pathway expansion, the specific RBPs (RNA binding proteina) regulating RNA functions may be potential therapeutic targets in their own right. RNP-IP is a technology that allows the isolation and identification of mRNAs, microRNAs and protein components of RNP complexes from cell extracts using antibodies to RBPs. Once purified, the RNAs present in the complex are analyzed to identify the target mRNAs using various molecular biology tools such as RT-PCR, gene expression analysis based on microarray technology (Chip analysis), or sequencing. Using this method, more RNA that is present in the nucleus can be obtained.

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[Abstract]
Post-transcriptional regulation of gene expression is a ribonucleoprotein (RNP)-driven process, which involves RNA binding proteins (RBPs) and noncoding RNAs that regulate splicing, nuclear export, subcellular localization, mRNA stability and translation. mRNAs encoding proteins that function in a particular cell process or pathway can be found within a unique mRNP complex, which consists of mRNA and RNP. This provides valuable information regarding not only known components of a particular process or pathway, but importantly, leads to the identification of novel components representing potential therapeutic targets and biomarkers. In addition to those targets identified by pathway expansion, the specific RBPs (RNA binding proteina) regulating RNA functions may be potential therapeutic targets in their own right. RNP-IP is a technology that allows the isolation and identification of mRNAs, microRNAs and protein components of RNP complexes from cell extracts using antibodies to RBPs. Once purified, the RNAs present in the complex are analyzed to identify the target mRNAs using various molecular biology tools such as RT-PCR, gene expression analysis based on microarray technology (Chip analysis), or sequencing. Using this method, more RNA that is present in the nucleus can be obtained.

Materials and Reagents

Normal Rabbit IgG

High-salt solution

RIP-certified antibody (NBL, catalog number depends on what do you want to target)

Preparation of Quality Check (QC) sample In order to confirm whether RIP-Assay is running properly, we recommend to perform quality check. Collect QC sample and check the protein and RNA expression level at some steps. At least two additional aliquots may be retained for quality check. Use one of the aliquots (10 μl of precleared cell lysate, Input sample) for analysis of RBP expression level by Western Blotting, and use other aliquots (10 μl of precleared cell lysate) for analysis of Total RNA (See Example of RIP-Assay Results).

Resolve 20 μl of the prepared sample on SDS-PAGE, and proceed to western blotting analysis.

Preparation of total RNA (for quality check of Total RNA)

Place 10 μl of precleared cell lysate at -80 °C until beginning of RNA isolation.

After RNP immunoprecipitation, use the lysate to prepare Total RNA sample according to RNA isolation protocol (See below).

Transfer 500 μl of the precleared cell lysate to the tube (prepared in step 33) containing Antibody-immobilized protein A or protein G agarose beads washed once with lysis buffer (+), that was prepared in steps 30-33.

For fourth wash, add 1 ml of wash buffer (+), then mix well and dispense 100 μl of the mixture to new microcentrifuge tube for QC sample (post-IP beads). Use those aliquots for quality check by western blotting (See Example of RIP-Assay Results).

Resolve 20 μl of the prepared sample on SDS-PAGE, and proceed to western blotting analysis.

Lysis buffer
Add appropriate concentrations of protease inhibitors, RNase inhibitor, and DTT to lysis buffer just before use. Lysis buffer containing these reagents is described as lysis buffer (+) in the following protocols. The optimal concentration of each reagent for RIP-Assay is shown as follows.

Precaution: Additional buffer preparation
In some cases, both the lysis buffer (+) and wash buffer (+) may require the addition of appropriate volumes of high-salt solution (in these cases, add 30 μl of high-salt solution to each ml of lysis buffer and wash buffer).

RNA isolation (from Antibody-immobilized protein A or protein G agarose beads-RNP complex) Solution II and Solution III should be equilibrated to room temperature before use. Reagents should be briefly but thoroughly mixed before use.

Prepare master mix solution by diluting 10 μl of Solution I with 390 μl of Solution II per sample.

Dispense 2 μl of Solution IV to each new microcentrifuge tube for step 5.

Add 250 μl of Solution III to each tube, vortex thoroughly, then centrifuge the tube at 2,000•x g for 2 min at RT.

Carefully transfer the supernatant to the tube containing 2 μl of Solution IV prepared in step 2 (avoid removing the protein A or protein G agarose beads from the pellet. Contamination of the beads may affect following steps).

Add 600 μl of ice-cold 2-propanol to each tube, vortex briefly but thoroughly, then spin-down.

Incubate the tube at -20 °C or below for 20 min (or for overnight, if necessary).

Centrifuge the tube at 12,000 x g for 10 min at 4 °C, then aspirate the supernatant carefully.

Rinse the pellet with 500 μl of ice-cold 70% ethanol, and mix briefly.

Centrifuge the tube at 12,000 x g for 3 min at 4 °C, then aspirate the supernatant carefully.

Rinse the pellet once again using steps 9-10.

Dry up the pellet by aspirating excess ethanol followed by evaporation for 5-15 min at RT. Avoid RNase contamination (evaporation in clean bench is recommended).

Reconstitute the pellet in 20 μl of nuclease-free water.

Store at -80 °C until starting following analysis.

Acknowledgments

This protocol was adapted from the NBL RIP-Assay Kit (see Reference 1).

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I only used cell lysates to get the target protein. You know the principle of this method is to use specificity of Ag-Ab reaction. If your target protein is outside of the cells, you should think about it amount is enough or not for this method. From theory, it is feasible. the problem is how do you extract the raw protein products. If you use other biochemical methods to purify the target protein , then run RIP, it may be better.

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You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.